Anthony J. Kim

Self-Assembly of Janus Dendrimers into Uniform Dendrimersomes and Other Complex Architectures

Janus Drug Delivery Vehicle Efficient drug delivery vehicles need to be produced in a limited size range and with uniform size distribution. The self-assembly of traditional small-molecule and polymeric amphiphiles has led to the production of micelles, liposomes, polymeric micelles, and polymersomes for use in drug delivery applications. Now, Percec et al. (p. 1009) describe the self-assembly of Janus-type (i.e., two-headed) dendrimers to produce monodisperse supramolecular constructs, termed “dendrimersomes,” and other complex architectures. The structures, which showed long-term stability as well as very narrow size distributions, were easily produced by the injection of an ethanolic solution of the dendrimer into water. The dendrimersomes could be loaded with the anticancer drug doxorubicin and exhibited controlled drug release with changing pH. Amphiphilic, spherically shaped polymers self-assemble into larger hollow complexes that could be used for drug delivery. Self-assembled nanostructures obtained from natural and synthetic amphiphiles serve as mimics of biological membranes and enable the delivery of drugs, proteins, genes, and imaging agents. Yet the precise molecular arrangements demanded by these functions are difficult to achieve. Libraries of amphiphilic Janus dendrimers, prepared by facile coupling of tailored hydrophilic and hydrophobic branched segments, have been screened by cryogenic transmission electron microscopy, revealing a rich palette of morphologies in water, including vesicles, denoted dendrimersomes, cubosomes, disks, tubular vesicles, and helical ribbons. Dendrimersomes marry the stability and mechanical strength obtainable from polymersomes with the biological function of stabilized phospholipid liposomes, plus superior uniformity of size, ease of formation, and chemical functionalization. This modular synthesis strategy provides access to systematic tuning of molecular structure and of self-assembled architecture.

Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier

Inhaled gene carriers must penetrate the highly viscoelastic and adhesive mucus barrier in the airway in order to overcome rapid mucociliary clearance and reach the underlying epithelium; however, even the most widely used viral gene carriers are unable to efficiently do so. We developed two polymeric gene carriers that compact plasmid DNA into small and highly stable nanoparticles with dense polyethylene glycol (PEG) surface coatings. These highly compacted, densely PEG-coated DNA nanoparticles rapidly penetrate human cystic fibrosis (CF) mucus ex vivo and mouse airway mucus ex situ. Intranasal administration of the mucus penetrating DNA nanoparticles greatly enhanced particle distribution, retention and gene transfer in the mouse lung airways compared to conventional gene carriers. Successful delivery of a full-length plasmid encoding the cystic fibrosis transmembrane conductance regulator protein was achieved in the mouse lungs and airway cells, including a primary culture of mucus-covered human airway epithelium grown at air-liquid interface, without causing acute inflammation or toxicity. Highly compacted mucus penetrating DNA nanoparticles hold promise for lung gene therapy.

Use of Single-Site-Functionalized PEG Dendrons To Prepare Gene Vectors that Penetrate Human Mucus Barriers†

Protective mucus layers serve as the body’s first line of defense at exposed surfaces of the eyes and respiratory, gastrointestinal, and cervicovaginal tracts. These highly viscoelastic and adhesive mucus gels trap most foreign pathogens and environmental ultrafine particles, which are then removed by mucus clearance mechanisms[1] (on the order of seconds to a few hours, depending on anatomical site). However, mucus also immobilizes and rapidly clears therapeutic nanoparticles, including synthetic drug carriers,[2] and clinically tested viral[3] and nonviral gene vectors[4] and, therefore, represents a critical obstacle to localized drug and gene delivery at mucosal surfaces for the treatment of a variety of diseases.[1b]

Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.

Enhancement of airway gene transfer by DNA nanoparticles using a pH-responsive block copolymer of polyethylene glycol and poly-l-lysine

Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-L-lysine (PEG-CK(30)), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-L-histidine was inserted between PEG and poly-L-lysine to form a triblock copolymer system, PEG-CH(12)K(18). Inclusion of poly-L-histidine increased the buffering capacity of PEG-CH(12)K(18) to levels comparable with branched polyethyleneimine. PEG-CH(12)K(18) compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK(30) DNA nanoparticles. PEG-CH(12)K(18) DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH(12)K(18) DNA nanoparticles improved the in vitro gene transfer by ~20-fold over PEG-CK(30) DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ~3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles.

Non-degradative intracellular trafficking of highly compacted polymeric DNA nanoparticles

Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-l-lysine via a single cysteine residue (CK(30)PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK(30)PEG DNPs made using two different molecular weights of PEG, CK(30)PEG(10k) and CK(30)PEG(5k). We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2h, 37±19% and 47±8% for CK(30)PEG(10k) and CK(30)PEG(5k), respectively. CK(30)PEG(10k) and CK(30)PEG(5k) DNPs moved within live cells at average velocities of 0.09±0.04μm/s and 0.11±0.04μm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.

Minimizing the non-specific binding of nanoparticles to the brain enables active targeting of Fn14-positive glioblastoma cells.

A major limitation in the treatment of glioblastoma (GBM), the most common and deadly primary brain cancer, is delivery of therapeutics to invading tumor cells outside of the area that is safe for surgical removal. A promising way to target invading GBM cells is via drug-loaded nanoparticles that bind to fibroblast growth factor-inducible 14 (Fn14), thereby potentially improving efficacy and reducing toxicity. However, achieving broad particle distribution and nanoparticle targeting within the brain remains a significant challenge due to the adhesive extracellular matrix (ECM) and clearance mechanisms in the brain. In this work, we developed Fn14 monoclonal antibody-decorated nanoparticles that can efficiently penetrate brain tissue. We show these Fn14-targeted brain tissue penetrating nanoparticles are able to (i) selectively bind to recombinant Fn14 but not brain ECM proteins, (ii) associate with and be internalized by Fn14-positive GBM cells, and (iii) diffuse within brain tissue in a manner similar to non-targeted brain penetrating nanoparticles. In addition, when administered intracranially, Fn14-targeted nanoparticles showed improved tumor cell co-localization in mice bearing human GBM xenografts compared to non-targeted nanoparticles. Minimizing non-specific binding of targeted nanoparticles in the brain may greatly improve the access of particulate delivery systems to remote brain tumor cells and other brain targets.

Evolving Drug Delivery Strategies to Overcome the Blood Brain Barrier.

The blood-brain barrier (BBB) poses a unique challenge for drug delivery to the central nervous system (CNS). The BBB consists of a continuous layer of specialized endothelial cells linked together by tight junctions, pericytes, nonfenestrated basal lamina, and astrocytic foot processes. This complex barrier controls and limits the systemic delivery of therapeutics to the CNS. Several innovative strategies have been explored to enhance the transport of therapeutics across the BBB, each with individual advantages and disadvantages. Ongoing advances in delivery approaches that overcome the BBB are enabling more effective therapies for CNS diseases. In this review, we discuss: (1) the physiological properties of the BBB, (2) conventional strategies to enhance paracellular and transcellular transport through the BBB, (3) emerging concepts to overcome the BBB, and (4) alternative CNS drug delivery strategies that bypass the BBB entirely. Based on these exciting advances, we anticipate that in the near future, drug delivery research efforts will lead to more effective therapeutic interventions for diseases of the CNS.

The TWEAK receptor Fn14 is a potential cell surface portal for targeted delivery of glioblastoma therapeutics

Fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is the cell surface receptor for the tumor necrosis factor (TNF) family member TNF-like weak inducer of apoptosis (TWEAK). The Fn14 gene is normally expressed at low levels in healthy tissues but expression is significantly increased after tissue injury and in many solid tumor types, including glioblastoma (GB; formerly referred to as ‘GB multiforme’). GB is the most common and aggressive primary malignant brain tumor and the current standard-of-care therapeutic regimen has a relatively small impact on patient survival, primarily because glioma cells have an inherent propensity to invade into normal brain parenchyma, which invariably leads to tumor recurrence and patient death. Despite major, concerted efforts to find new treatments, a new GB therapeutic that improves survival has not been introduced since 2005. In this review article, we summarize studies indicating that (i) Fn14 gene expression is low in normal brain tissue but is upregulated in advanced brain cancers and, in particular, in GB tumors exhibiting the mesenchymal molecular subtype; (ii) Fn14 expression can be detected in glioma cells residing in both the tumor core and invasive rim regions, with the maximal levels found in the invading glioma cells located within normal brain tissue; and (iii) TWEAK:Fn14 engagement as well as Fn14 overexpression can stimulate glioma cell migration, invasion and resistance to chemotherapeutic agents in vitro. We also discuss two new therapeutic platforms that are currently in development that leverage Fn14 overexpression in GB tumors as a way to deliver cytotoxic agents to the glioma cells remaining after surgical resection while sparing normal healthy brain cells.

Emerging Applications of Therapeutic Ultrasound in Neuro-oncology: Moving Beyond Tumor Ablation.

: Transcranial focused ultrasound (FUS) can noninvasively transmit acoustic energy with a high degree of accuracy and safety to targets and regions within the brain. Technological advances, including phased-array transducers and real-time temperature monitoring with magnetic resonance thermometry, have created new opportunities for FUS research and clinical translation. Neuro-oncology, in particular, has become a major area of interest because FUS offers a multifaceted approach to the treatment of brain tumors. FUS has the potential to generate cytotoxicity within tumor tissue, both directly via thermal ablation and indirectly through radiosensitization and sonodynamic therapy; to enhance the delivery of therapeutic agents to brain tumors by transiently opening the blood-brain barrier or improving distribution through the brain extracellular space; and to modulate the tumor microenvironment to generate an immune response. In this review, we describe each of these applications for FUS, the proposed mechanisms of action, and the preclinical and clinical studies that have set the foundation for using FUS in neuro-oncology. ABBREVIATIONS BBB, blood-brain barrierCED, convection-enhanced delivery5-Ala, 5-aminolevulinic acidFUS, focused ultrasoundGBM, glioblastoma multiformeHSP, heat shock proteinMRgFUS, magnetic resonance-guided focused ultrasoundpFUS, pulsed focused ultrasound.